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PubMed1989

A serum protein SP40,40 modulates the formation of membrane attack complex of complement on erythrocytes.

SP40,40 was isolated from the soluble membrane attack complex (SC5b-9) by HPLC using a reverse-phase column. Amino acid compositions of its alpha- and beta-subunits were similar to each other with the exception of glycine content. Amino-terminal sequences of its alpha- and beta-subunits were identical to those of the subunits prepared from human serum, respectively, indicating that there was no degradation of SP40,40 during incorporation into SC5b-9. When guinea pig erythrocytes were incubated with C56f, C7, C8 and C9 in the presence of SP40,40, SP40,40 enhanced the hemolysis. The protein, however, inhibited the hemolysis when erythrocytes were pre-incubated with C56f and SP40,40 prior to the addition of C7, C8 and C9. These findings indicate that SP40,40 modulates the formation of membrane attack complex by interacting with C56f at the first step, and that the co-existence of other factors, besides C56f, is required for the enhancing activity of SP40,40.

Amino Acids↗

PubMed1988

The cytolytic C5b-9 complement complex: feedback inhibition of complement activation.

We describe a regulatory function of the terminal cytolytic C5b-9 complex [C5b-9(m)] of human complement. Purified C5b-9(m) complexes isolated from target membranes, whether in solution or bound to liposomes, inhibited lysis of sensitized sheep erythrocytes by whole human serum in a dose-dependent manner. C9 was not required for the inhibitory function since C5b-7 and C5b-8 complexes isolated from membranes were also effective. No effect was found with the cytolytically inactive, fluid-phase SC5b-9 complex. However, tryptic modification of SC5b-9 conferred an inhibitory capacity to the complex, due probably to partial removal of the S protein. Experiments using purified components demonstrated that C5b-9(m) exerts a regulatory effect on the formation of the classical- and alternative-pathway C3 convertases and on the utilization of C5 by cell-bound C5 convertases. C5b-9(m) complexes were unable to inhibit the lysis of cells bearing C5b-7(m) by C8 and C9. Addition of C5b-9(m) to whole human serum abolished its bactericidal effect on the serum-sensitive Escherichia coli K-12 strain W 3110 and suppressed its hemolytic function on antibody-sensitized, autologous erythrocytes. Feedback inhibition by C5b-9(m) represents a biologically relevant mechanism through which complement may autoregulate its effector functions.

Complement Activation↗

PubMed1988

Biochemical characterization of the human complement protein C6. Association with alpha-thrombin-like enzyme and absence of serine protease activity in cytolytically active C6.

Complement protein C6 has been proposed by others to be a serine protease whose activity is obligatory for complement-directed cell lysis. We separated the serine protease (Mr approximately 30,000) activity found associated with apparently homogeneous preparations of C6 from the hemolytically active C6 protein. The protease was characterized as thrombin-like based on substrate specificity, inhibitor profile, and kinetic studies. Although the proteolytic activity of C6 preparations was inhibitable by several inhibitors of serine proteases, the C6 hemolytic activity remained unaffected. Acid-induced (C(5,6)a complex formation between C5 and C6 (protease-free) was demonstrated by ion-exchange fast protein liquid chromatography, reversed-phase high performance liquid chromatography, and reactive cytolytic activity in the presence of C7, C8, and C9; but no cleavage of the alpha-chain of C5 was observed. Diisopropylphosphorofluoridate pretreatment of the components did not affect their ability to generate functionally active (C(5,6)a. Evidently, C6-associated thrombin is not required for formation of functional C(5,6)a. Thus, C6 does not function in the membrane attack pathway of complement as a serine protease. A method for the isolation of homogeneous C6 in the hemolytically fully active form is described. No free sulfhydryl group was detected in C6. The amino acid sequence of 20 amino-terminal residues was determined.

Amino Acid Sequence↗

PubMed1975

Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule. III. Further evidence that complement-mediated cell lysis is involved.

The results of our previous studies have suggested that serum-induced inhibition of proximal tubular fluid absorption is due to complement-induced lysis of the tubular cells. The present study provides further evidence in support of this idea as well as other information pertinent to the mechanism of complement activation in vivo. 1. The electrical resistance of the luminal brush border membrane is reduced drastically concomitantly with a drop in cell potential difference when serum is perfused intraluminally. 2. Human C1 inhibitor (30-50 units/ml) does not significantly affect the inhibitory activity of human serum on fluid absorption, suggesting the non-involvement of the classical pathway. 3. Reactive lysis reagents (C56, C7, C8 + C9) partially inhibit fluid absorption when infused into the lumen. 4. In contrast to our previous report (Sato, K. and Ullrich, K.J. (1974) Biochim. Biophys. Acta 354, 182-187), very fresh serum, 10-times diluted can inhibit fluid absorption if perfused for 10 min. 5. Both mouse and guinea pig serum, which are normally inactive, are activated to attack the tubular cells if 1/100 volume rat or rabbit serum is added to them No such activation occurs by mixing guinea pig serum and mouse serum. The available data suggest that the presence of the later complement components but not the heat-labile factor (Factor B) or C3PA or C1 in the added serum is a prerequisite for mouse and guinea pig sera to be activated to inhibit fluid absorption.

Animals↗

PubMed1988

Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels.

A protein which inhibited complement channel formation was isolated from extracts of papain-digested human erythrocyte membranes using DEAE-Sephacel, Bio-Gel A0.5m column chromatographies, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper and elution with 2% NP-40 solution. The purified protein showed a molecular weight of 18 kDa, and efficiently inhibited hemolysis of EC5-7 cells with C8 and C9, but did not show any decay-accelerating activity to C5 convertase. Immunochemical analysis of native membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the antibody against this protein gave a single band having the same mobility as this protein; papain did not eliminate a significant portion of this protein.

Chromatography, DEAE-Cellulose↗

PubMed1987

Inherited deficiencies of complement components in man.

Isolated inherited deficiency states of almost every complement protein have been recognized. Almost all are autosomal recessive traits. Deficiency of the early-acting components C1, C4 and C2 is associated with increased risk of immune complex disease, particularly systemic lupus erythematosus. Patients with deficiency of C3, factor I or factor H have increased susceptibility to infection by pyogenic bacteria, whereas those with deficiencies of properdin, C5, C6, C7 or C8 are prone to systemic neisserial infection. Inherited deficiency of C1 inhibitor is transmitted as an autosomal dominant trait, is genetically heterogeneous, and is associated with attacks of angioedema and consumption of C4 and C2. There is evidence that a plasmin-modified fragment of C2 is responsible for the angioedema in this disorder. Administration of androgens tends to correct the biochemical abnormalities of hereditary angioedema and to prevent attacks.

Angioedema↗

PubMed1992

Activation of the fifth component of human complement, C5, without cleavage, by methionine oxidizing agents.

Purified human C5 was incubated with chloramine T (Cl-T) or N-chloro-succinimide (N-Cl-S) in barbital buffer, pH 7.2. The treatment led to C5 activation: Cl-T- and N-Cl-S-treated C5 acquired a binding site for C6; upon incubation with C6 and subsequent addition of C7, C8 and C9 a membrane attack complex formed which lysed non-sensitized guinea pig red cells (reactive lysis). While the physiological activation of C5 follows its specific cleavage, the resulting fragment C5b representing the activated C5 and expressing the C6 binding site, the treatment with the mentioned chemicals does not lead to fragmentation of the C5 protein. So, functionally, the product of the chemical treatment is C5b-like, but chemically, it comprises the whole protein; no C5a is released. Cl-T and N-Cl-S are known to more or less selectively oxidize methionine residues in proteins, dependent on the conditions. Other sensitive amino acid residues are tryptophan and cysteine. Conditions were chosen for treatment of C5 with Cl-T which exclude attack on tryptophan, and we have ensured that human C5 does not contain free cysteine residues. Further, oxidation of about 60% of the methionine residues of C5 by Cl-T was demonstrated by amino acid analysis. So, all evidence points to methionine residue(s) as the site of attack of Cl-T and probably also of N-Cl-S. The oxidation product of methionine, its sulphoxide, may cause a change in structural conformation of C5 which involves expression of the C6 binding site. Earlier it was found that oxidation of C5 by hydroxyl radicals leads to its activation without cleavage. Since the properties of this C5b-like product resemble those of the product of treatment with Cl-T and N-Cl-S, it is suggested that the formerly found activation of human C5 by hydroxyl radicals is also mediated by oxidation of methionine residue(s) in the C5 protein.

Chloramines↗

PubMed1986

An enzyme-linked immunoabsorbent assay for the quantitation of the terminal complement complex from cell membranes or in activated human sera.

A sensitive and simple enzyme-linked immunoabsorbent assay (ELISA) has been developed to measure the terminal complement complex (TCC) in solution. Commercially available antibodies to the native complement (C) components C5 and C9 were used in a double antibody sandwich technique sensitive enough to detect 0.3 microgram/ml of purified TCC. The TCC was not detected in normal human serum (NHS) nor was it generated when sera from patients with a genetic deficiency of functional C5, C7, C8 beta or C9 were activated with cobra venom factor (CVF). If the C8 beta deficient serum was reconstituted with the C8 beta chain and incubated with CVF, TCC were formed and detected by the assay. In in vitro experiments, the TCC was detected in NHS activated by either the classical or alternative pathway even when there was no measurable consumption of C5, C8 or C9. In addition, adaptation of a detergent extraction procedure permitted the quantitation by the assay, of TCC which were generated on sensitized sheep erythrocyte membranes. Experiments to test sample handling conditions showed no generation of TCC in NHS after four freeze/thaw cycles and spontaneous formation only if NHS had been incubated at 37 degrees C for 48 h. The TCC in zymosan-activated NHS were stable at 37 degrees C for 1 week. Patients with C activation associated diseases such as SLE and rheumatoid arthritis had increased levels of TCC that correlated with positive clinical tests for inflammation, even though C levels were normal when measured by routine techniques. These results suggest that this ELISA will provide a valuable tool for studying the role of C in the pathogenesis of C-mediated diseases and in examining the mechanism of tissue injury in in vitro experimental systems.

Animals↗

PubMed1983

Consequences of cell membrane attack by complement: release of arachidonate and formation of inflammatory derivatives.

Treatment of [3H]arachidonic acid [( 3H]C20:4)-labeled and antibody-sensitized Ehrlich ascites tumor cells with guinea pig or rabbit serum complement (C) released up to about 20 or 25% of the incorporated [3H]C20:4 into the aqueous phase as a consequence of C-induced hydrolysis of cellular phospholipid. The dose-response curve of release of [3H]C20:4 from Ehrlich ascites tumor cells, with respect to C, was approximately in the same range as the cytolytic response. In the case of [3H]C20:4-labeled and antibody-sensitized peritoneal mouse macrophages, treatment with C induced release of about 11% of the incorporated 3H as C20:4 and about 6% as prostaglandins, thromboxane B2, and hydroxyicosatetraenoic acids. C6- and C8-deficient rabbit and human sera, respectively, induced release of small amounts of [3H]C20:4 from Ehrlich ascites tumor cells and macrophages; these deficient sera also released traces of oxygenated derivatives from macrophages. Addition of purified C6 or C8 effectively restored release from both cell types, indicating that the terminal C proteins, up to and including C8, are required for the major part of the release. Our results do not rule out a possible requirement for C9.

Animals↗

PubMed2006

Sphingomyelinases D induce direct association of C1q to the erythrocyte membrane causing complement mediated autologous haemolysis.

Bites by Loxosceles spiders can induce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis and persistent inflammation. The causative toxin is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have previously found that SMase D toxins led to an increased susceptibility of human erythrocytes (E) to activation of complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the haemolysis induced by SMases from Corynebacterium pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). When P1 or PLD treated E were incubated with C8-depleted human serum, an increase in C1q, serum amyloid protein (SAP) and C-reactive protein (CRP) binding was observed. While purified C1q, SAP and CRP were found to bind to P1 or PLD treated E, depletion of SAP or CRP from human serum did not prevent C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation. However depletion of C1 lead to a greatly reduced haemolysis, demonstrating that the activation of the CP is caused by direct binding of C1q to the SMase treated cells. Binding of fluid phase C-regulators C4b-binding protein and factor H was also observed, however these C-regulators in conjunction with the membrane bound C-regulators were unable to prevent haemolysis, demonstrating the potency of SMase D facilitated binding of C1 and activation of C.

Animals↗

PubMed1990

Fluid phase generation of terminal complement complex as a novel index of bioincompatibility.

Blood membrane interactions in hemodialysis have been shown to trigger complement (C) activation. As indicators of C-activation the anaphylatoxins (C3a and C5a) are problematical because of methodological difficulties and their kinetic properties. We developed a sensitive and specific micro-ELISA using a monoclonal antibody against neoantigens on the terminal complement complex (TCC); highly purified human TCC served as standard. Concentrations of TCC were measured in single-path perfusion systems (in vitro) and in the blood lines (arterial inlet; venous outlet) of patients on hemodialysis using steam-sterilized or ETO-sterilized dialyzers with the following membranes: cuprophan (CU), hemophan (HE) and polysulfone F6 (PS), respectively. All dialyzers with identical geometry were run under identical conditions. All membranes tested caused continuously ongoing net generation of TCC. In vitro, contact of serum with CU minidialyzers resulted in fivefold higher net release of TCC compared with HE and PS. In vivo TCC concentration-time profiles differed significantly between membranes in the rank order CU much much greater than HE greater than PS (mean basal concentration 58 x 10(-11) M; peak increase over baseline with CU 40-fold, HE fourfold, PS threefold). In addition, more TCC was generated from the same dialyzers with ETO than steam sterilization. TCC differed from C3a and C5a in the following respects: (i) lower detection limit (4 x 10(-11) vs. less than 5 x 10(-9) M for both C-anaphylatoxins); (ii) higher relative increment (inlet) during CU dialysis (25-fold vs. eightfold and twofold, respectively); (iii) C-anaphylatoxins yielded the same ranking (CU much greater than HE greater than PS), but TCC concentrations were not a linear function of C3a or C5a concentrations, respectively. Kinetic analysis (Bateman function) showed significant differences of invasion constants between membranes, that is, CU 0.088 min-1, HE 0.09, PS 0.168. The net amount of TCC released from the dialyzer was calculated under certain assumptions. It was 75.5 mg/4 hr for CU, 7.3 for HE and 5.0 for PS. The elimination constant was also dependent on the type of membrane. Using flow cytofluorometry and immunohistochemical methods (APAAP), TCC was demonstrated on membranes of granulocytes obtained during dialysis; this is compatible with potential in vivo cell activation. Generation of PGE2 and TNF alpha by adherent monocytes induced by cuprophan was C8 dependent: levels were significantly increased by addition of C8 to C8 deficient human serum concomitantly with generation of TCC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1976

Membrane fluidity change in erythrocytes induced by complement system.

The structural change in erythrocyte membranes induced by antibody and complement was studied using phospholipid spin-labels. Sheep erythrocytes were labeled with phosphatidylcholine spin-label and various intermediate cells (erythrocyte-antibody complex (EA), EA bound with complement components from C1 to C7 (EAC1-7), EAC1-8, and EAC1-9) were prepared. Electron spin resonance spectra of EA, EAC1-7, and EAC1-8 were very similar to that of the erythrocytes, while that of EAC1-9 was markedly different. The overall splitting value for the lysed EAC1-9 (53 G) was much smaller than that for the erythrocytes (57 G), indicating a marked fluidization around the phosphatidylcholine label. The unlysed EAC1-9 membranes contained a limited fraction of the fluidized area. When EA was reacted with complement in the presence of 36% bovine serum albumin, the membranes were fluidized similarly to the lysed EAC1-9, although the hemolysis was largely blocked. The membranes of unlysed EAC1-9 prepared in isotonic (ethylenedinitrilo)tetraacetic acid were also fluidized, but to somewhat smaller extent. The role of C9 in the modification of erythrocyte membranes was also demonstrated using Mg2+ ghosts, which were prepared by hypotonic hemolysis in the presence of Mg2+. The membranes of Mg2+ ghost of EAC1-7 were markedly fluidized when bound with C8 and C9, but not affected by binding of C8 only. The component C8 was found to give a latent effect on the membranes that caused irreversible fluidization upon osmotic shock. The terminal component thus creates a fluidized area in the erythrocyte membranes through which small ions and molecules may diffuse more easily and the resulting osmotic unbalance may finally cause hemolysis.

Animals↗

PubMed1997

Kinetics of DNA polymerase I (Klenow fragment exo-) activity on damaged DNA templates: effect of proximal and distal template damage on DNA synthesis.

Mutagenic DNA adducts have been analyzed with respect to the rate of nucleotide insertion opposite the modified base, extension from that "mispair", and nucleotide insertion preference. To complement and extend these studies we have investigated the long-range effects of DNA adducts on DNA polymerase activity. To address this question, primer extension reactions were performed using DNA polymerase I, Klenow fragment exo-. Templates containing 7,8-dihydro-8-oxoguanine, dG-C8-aminofluorene, dG-C8-(acetylamino)fluorene, and the model abasic site, tetrahydrofuran, were used for these studies, and the steady-state kinetics of correct nucleotide insertion were determined at positions (-2), (-1), (+1), (+2), (+3), and (+5) with respect to the template lesion. The kinetics of primer extension by Klenow fragment exo- at template positions 3' to the lesion showed only a small inhibitory effect, <3-fold, even for the strongly blocking lesion, dG-C8-(acetylamino)fluorene, indicating that Klenow fragment exo- activity is not greatly affected by lesions in the single-stranded portion of the template-primer. In contrast, a dramatic decrease in the frequency of primer extension was observed at template sites 5' to the site of adduction. Inhibition of polymerase activity decreased as the distance from the lesion increased; however, a relatively large effect was seen at the (+2) and (+3) positions for dG-C8-(acetylamino)fluorene and tetrahydrofuran. For these blocking lesions, the effect on extension 5 bases from the lesion was greatly reduced. We conclude from these studies that DNA damage at positions remote from the site of the lesion affects DNA polymerase function.

DNA Damage↗

PubMed1994

[A case of systemic lupus erythematosus in late component (C9) complement deficiency].

We report on a case of C9 deficiency followed by the onset of systemic lupus erythematosus (SLE). A 51-year-old female patient had suffered from repeated urinary tract infections since 1977. In 1980, at 41 years of age, she had proteinuria and facial erythema along with low level of CH50 less than 12.0 U/ml. She was diagnosed as C9 deficiency (C9D) because of C9 protein less than 0.5 mg/dl, C9 activity 0.03% and C8 activity 94% and also SLE was strongly suspicious. Subsequently, she suffered from repeated urinary tract infections. In 1990, she was diagnosed as SLE because of pleuritis, pericarditis and positive anti-nuclear (1:640) and positive anti-DNA antibody (28.0 U/ml) in addition to proteinuria and facial erythema. From the family study, her two sisters showed homozygous C9D (less than 0.5 mg/dl) with hypergammaglobulinemia (2290 and 2230 mg/dl, respectively) and positive anti-nuclear antibody (1:80) in both. Her father also showed heteropygous C9D (1.7 mg/dl) with anti-nuclear antibody (1:640). These results suggest that healthy carriers of C9D may have some abnormalities in humoral or cell-mediated immunity. The possibility was thought that other unrecognized disease associated loci exist in linkage with the C9 allele like that of C4D was speculated. And repeated infections due to C9D may possibly induce B cell activation followed by the occurrence of SLE or related disorders. This case seems to be very valuable with respect to the onset of SLE under a long-term observation of C9D.

Complement C9↗

PubMed1989

A biotin-avidin sandwich ELISA for quantification of intact complement component C9. The sera from hereditary C9 deficient individuals completely lack C9.

A two-site sandwich ELISA method was developed for quantitating intact C9 protein using MoAb P40 (anti-C9b antibody). This antibody reacted with monomeric C9 but not with polymerized C9. MoAb P40 was used as a capture antibody and MoAb X195 (anti-C9a antibody) as a detection antibody. This method is highly sensitive and can detect approximately 0.5 ng/ml of native C9. No cross-reactivities of either C6, C7, or C8 were observed even at concentrations of 10 micrograms/ml per component. In addition, this method allows for measurement of only intact C9 molecules, eliminating the interference of polymerized C9 or inactivated C9. Using this assay, no C9 at all was detected in sera from inherited C9 deficient individuals, including both healthy blood donors and patients with meningococcal meningitis; although by hemolytic assay, C9 levels were reported to be less than 0.2% those of NHS. Therefore, this two-site sandwich ELISA method can replace the hemolytic assay, and is especially useful for measuring small amounts of C9 in serum.

Animals↗

PubMed1990

[Terminal complement complex (TTC) levels in plasma and urine from glomerular diseases: enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody against neoantigens of TCC].

In order to get monoclonal antibodies (MoAbs) against neoantigens of terminal complement complex. MoAbs after immunization of mice with polymerized human C9 were screened for reactivities against native and polymerized C9. MoAb 1B4 reacted with tubular C9 polymer, but did not react with either native or sodium dodecyl sulfate-denatured monomeric C9 as revealed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Moreover, MoAb 1B4 reacted with the terminal complement complex (TCC), that is, membrane attack complex and the fluid-phase SC5b-9 complex. Thus, MoAb 1B4 recognized a neoantigen in the moiety of C9 polymer in the TCC. Thereafter, we measured TCC in plasma and urine with sandwich ELISA using 1B4 and antihuman C7 antibody to evaluate terminal complement activation in patients with glomerular diseases. TCC was detectable in plasma but not in urine from most of normal controls. In plasma, TCC was elevated in 5 of 23 with lupus nephritis and in 6 of 11 with membranoproliferative glomerulonephritis, but all patients with IgA nephritis, focal glomerulosclerosis, membranous glomerulonephritis and minimal change lesions (MC) showed normal levels. In urine, TCC was detectable in most of patients with severe proteinuria (greater than or equal to 100 mg/dl) except MC. The TCC present in urine was partially purified by gel filtration with Sepharose 6B and was found to contain C5, C6, C7, C8, C9, and S protein by ELISA. Although the molecular weight of SC5b-9 complex is similar to IgM, fractional excretion rate of TCC was about 100 times higher than that of IgM. These results suggest that urinary TCC contains SC5b-9 complex like plasma TCC and is mostly derived from renal origin.

Adolescent↗

PubMed1990

The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9.

A human E membrane protein that inhibits lysis by the purified human C5b-9 proteins was isolated and characterized. After final purification, the protein migrated as an 18- to 20-kDa band by SDS-PAGE. Elution from gel slices and functional assay after SDS-PAGE (nonreduced) confirmed that all C5b-9 inhibitory activity of the purified protein resided in the 18- to 20-kDa band. Phosphatidylinositol-specific phospholipase C digestion of the purified protein abolished 50% of its C5b-9 inhibitory activity, and removed approximately 15% of the protein from human E. Western blots of normal and paroxysmal nocturnal hemoglobinuria E revealed an absence of the 18- to 20-kDa protein in the paroxysmal nocturnal hemoglobinuria E cells. The identity of this E protein with leukocyte Ag CD59 (P18, HRF20) was confirmed immunochemically and by N-terminal amino acid sequence analysis. A blocking antibody raised against the purified protein reacted with a single 18- to 20-kDa band on Western blots of human erythrocyte membranes. Prior incubation of human E with the F(ab) of this antibody increased subsequent lysis by the purified human C5b-9 proteins. Potentiation of C5b-9-mediated lysis was observed when erythrocytes were preincubated with this blocking antibody before C5b-9 assembly was initiated, or, when this antibody was added after 30 min, 0 degrees C incubation of C5b-8-treated E with C9. Chicken E incubated with purified CD59 were used to further characterize the mechanism of its C-inhibitory activity. Preincorporation of CD59 into these cells inhibited lysis by C5b-9, regardless of whether CD59 was added before or after assembly of the C5b-8 complex. When incorporated into the membrane, CD59 inhibited binding of 125I-C9 to membrane C5b-8 and reduced the extent of formation of SDS-resistant C9 polymer. The inhibitory effect of CD59 on 125I-C9 incorporation was most pronounced at near-saturating input of C9 (to C5b-8). By contrast, CD59 did not inhibit either C5b67 deposition onto the cell surface, or, binding of 125I-C8 to preassembled membrane C5b67. Taken together, these data suggest that CD59 exerts its C-inhibitory activity by limiting incorporation of multiple C9 into the membrane C5b-9 complex.

Antigens, Differentiation↗

PubMed1985

Synthesis of C1 inhibitor (C1-INA) by a human monocyte-like cell line, U937.

Human monocytes are known to synthesize many of the components of complement, including C1-INA. In this report we demonstrate that the human monocyte-like cell line U937 is also capable of synthesizing functional C1-INA. This was shown in several ways, including 1) incorporation of tritiated amino acids into antigenic C1-INA, immunoprecipitation, and detection by fluorography; 2) a sensitive ELISA, which allowed quantitation of antigenic C1-INA in cell lysates, and 3) a C2-dependent hemolytic assay in which the functional activity of U937 C1-INA was assayed. Data from the ELISA indicate that U937 cells contain between 2.1 to 12.8 ng of C1-INA per 1 X 10(6) cells. Furthermore, fluorescence-activated cell sorter analysis revealed that approximately 16% of U937 cells carry C1-INA as a surface bound antigen. Other proteins found to be synthesized by U937 cells include C1r, C8, and possibly alpha-2-macroglobulin. These results suggest that the U937 cell line could be a convenient and valuable model for the study of monocyte C1-INA synthesis and physiology.

Amino Acids↗